Prevention of thrombus formation and/or stabilization

ABSTRACT

The present invention relates to the use of at least one antibody and/or one inhibitor for inhibiting factor XII and preventing the formation and/or the stabilization of three dimensional thrombi. It also relates to a pharmaceutical formulation and the use of factor XII as an anti-thrombotic target.

The subject of the present invention is, in the most general aspect, theprevention of the formation and/or stabilization of three-dimensionalarterial or venous thrombi.

In particular the present invention relates to the use of at least oneantibody and/or one inhibitor for inhibiting factor XII activity andpreventing the formation and/or the stabilization of thrombi andthrombus growth. It also relates to a pharmaceutical formulation and theuse of factor XII as an anti-thrombotic target.

Vessel wall injury triggers sudden adhesion and aggregation of bloodplatelets, followed by the activation of the plasma coagulation systemand the formation of fibrin-containing thrombi, which occlude the siteof injury. These events are crucial to limit posttraumatic blood lossbut may also occlude diseased vessels leading to ischemia and infarctionof vital organs. In the waterfall or cascade model, blood coagulationproceeds by a series of reactions involving the activation of zymogensby limited proteolysis culminating in the fulminant generation ofthrombin, which converts plasma fibrinogen to fibrin and potentlyactivates platelets. In turn, collagen- or fibrin-adherent plateletsfacilitate thrombin generation by several orders of magnitude byexposing procoagulant phosphatidyl serine (PS) on their outer surfacewhich propagates assembly and activation of coagulation proteasecomplexes and by direct interaction between platelet receptors andcoagulation factors.

Two converging pathways for coagulation exist that are triggered byeither extrinsic (vessel wall) or intrinsic (blood-borne) components ofthe vascular system. The “extrinsic” pathway is initiated by the complexof the plasma factor VII (FVII) with the integral membrane proteintissue factor (TF), an essential coagulation cofactor that is absent onthe luminal surface but strongly expressed in subendothelial layers ofthe vessel. TF expressed in circulating microvesicles might alsocontribute to thrombus propagation by sustaining thrombin generation onthe surface of activated platelets.

The “intrinsic” or contact activation pathway is initiated when factorXII (FXII, Hageman factor) comes into contact to negatively chargedsurfaces in a reaction involving high molecular weight kininogen andplasma kallikrein. FXII can be activated by macromolecular constituentsof the subendothelial matrix such as glycosaminoglycans and collagens,sulfatides, nucleotides and other soluble polyanions ornon-physiological material such as glass or polymers. One of the mostpotent contact activators is kaolin and this reaction serves as themechanistic basis for the major clinical clotting test, the (activated)partial thromboplastin time (PTT, aPTT). In reactions propagated byplatelets, activated FXII then activates FXI and FXIa in turn activatesfactor IX. Despite its high potency to induce blood clotting in vitro,the (patho)physiological significance of the FXII-triggered intrinsiccoagulation pathway is questioned by the fact that hereditary deficiencyof FXII as well as of high molecular weight kininogen and plasmakallikrein is not associated with bleeding complications. Together withthe observation that humans and mice lacking extrinsic pathwayconstituents, such as TF, FVII or factor IX, suffer from severe bleedingthis has lead to the current hypothesis that fibrin formation is in vivoexclusively initiated by the extrinsic cascade (Mackman, N. (2004). Roleof tissue factor in hemostasis, thrombosis, and vascular development.Arterioscler. Thromb. Vasc. Biol. 24, 1015-1022).

Like all physiological mechanisms, the coagulation cascade can becomeactivated inappropriately and result in the formation of haemostaticplugs inside the blood vessels. Thereby, vessels can become blocked andthe blood supply to distal organs limited. This process is known asthromboembolism and is associated with high mortality. In addition, theuse of prosthetic devices that are in contact with blood is severelylimited because of activation of the coagulation cascade and coating ofthe prosthetic surface, often compromising its function. Examples ofsuch prosthetic devices are haemodialysers, cardiopulmonary by-passcircuits, vascular stents and in-dwelling catheters. In cases where suchdevices are used, anticoagulants, such as heparin, are used to preventfibrin from depositing on the surface. However, some patients areintolerant of heparin, which can cause heparin induced thrombocytopenia(HIT) resulting in platelet aggregation and life threatening thrombosis.Furthermore, an intrinsic risk of all anticoagulants used in clinics isan associated increased risk of serious bleeding. Therefore, a need fornew types of anticoagulant exists that is not associated with suchcomplications and that can be used in affected patients or as superiortherapy concept preventing thrombosis without increased bleedingtendencies.

Hence, it is apparent that there still exists a need for an improvedmedication for the treatment or prophylaxis of thrombosis and similardisorders. Therefore, it is an object of the present invention tosatisfy such a need. For more than five decades it has been known thatdeficiency of coagulation factor XII is not associated with increasesspontaneous or injury related bleeding complications (Ratnoff, O. D. &Colopy, J. E. (1955) A familial hemorrhagic trait associated with adeficiency of a clot-promoting fraction of plasma. J Clin Invest 34,602-13). Indeed, although presenting a pathological aPTT (a clinicalclotting test that addresses the intrinsic pathway of coagulation)humans that are deficient in FXII do not suffer from abnormal bleedingeven during major surgical procedures (Colman, R. W. Hemostasis andThrombosis. Basic principles & clinical practice (eds. Colman R. W.,Hirsch. J., Mader V. J., Clowes A. W., & George J.) 103-122 (LippincottWilliams & Wilkins, Philadelphia, 2001). In contrast, deficiency of FXIIhad been associated with increased risk of venous thrombosis (Kuhli, C.,Scharrer, I., Koch, F., Ohrloff, C. & Hattenbach, L. O. (2004) FactorXII deficiency: a thrombophilic risk factor for retinal vein occlusion.Am. J. Ophthalmol. 137, 459-464., Halbmayer, W. M., Mannhalter, C.,Feichtinger, C., Rubi, K. & Fischer, M. (1993) Factor XII (Hagemanfactor) deficiency: a risk factor for development of thromboembolism.Incidence of factor XII deficiency in patients after recurrent venous orarterial thromboembolism and myocardial infarction. Wien. Med.Wochenschr. 143, 43-50.). Studies and case reports supporting this idearefer to the index case for FXII deficiency, Mr. John Hageman, who diedof pulmonary embolism. The hypothesis that FXII deficiency is associatedwith an increased prothrombotic risk is challenged by a recentreevaluation of several case reports linking FXII deficiency withthrombosis (Girolami, A., Randi, Gavasso, S., Lombardi, A. M. & Spiezia,F. (2004) The Occasional Venous Thromboses Seen in Patients with Severe(Homozygous) FXII Deficiency are Probably Due to Associated RiskFactors: A Study of Prevalence in 21 Patients and Review of theLiterature. J. Thromb. Thrombolysis 17, 139-143). In most cases theauthors identified concomitant congenital or acquired prothrombotic riskfactors in combination with factor FXII deficiency that could beresponsible for the thrombotic event independently of FXII. The largestepidemiological studies using well characterized patients (Koster, T.,Rosendaal, F. R., Briet, E. & Vandenbroucke, J. P. (1994) John Hageman'sfactor and deep-vein thrombosis: Leiden thrombophilia Study. Br. J.Haematol. 87, 422-424) and FXII-deficient families (Zeerleder, S. et al.(1999) Reevaluation of the incidence of thromboembolic complications incongenital factor XII deficiency-a study on 73 subjects from 14 Swissfamilies. Thromb. Haemost. 82, 1240-1246) indicated that there is nocorrelation of FXII deficiency and any pro- or anti-thrombotic risk.

Surprisingly and in contrast to common believe of those skilled in theart the applicant has discovered that the factor XII-driven intrinsiccoagulation pathway is essential for arterial thrombus formation in vivobut not necessary for normal tissue-specific hemostasis. Unexpectedly,these results change the long-standing concept that blood clotting invivo is exclusively mediated by the extrinsic pathway and place factorXII in a central position in the process of pathological thrombusformation.

Accordingly, the first subject of the invention is the use of at leastone antibody and/or at least one inhibitor for inhibiting factor XII andpreventing the formation and/or the stabilization of three-dimensionalarterial or venous thrombi. The anti-FXII antibody respective inhibitormay hereby function so as inhibiting the activation of FXII and/orinterfere with other portions of the FXII molecule that are criticallyinvolved in FXII activation.

Together with the fact that the intrinsic pathway is not required forhemostasis, this establishes factor XII as a promising new target forpowerful antithrombotic therapy. In addition these results are importantfor the development of anti-FXII agents to control other contactsystem-linked (patho)mechanisms such as inflammation, complementactivation, fibrinolysis, angiogenesis and kinin formation.

Therefore, the present invention further provides the use of such anantibody and/or inhibitor in the treatment or prophylaxis of a conditionor disorder related to arterial thrombus formation, i. e. stroke ormyocardial infarction, inflammation, complement activation,fibrinolysis, angiogenesis and/or diseases linked to pathological kininformation such as hypotonic shock, edema including hereditaryangioedema, bacterial infections, arthritis, pancreatitis, or articulargout.

In particular, the use of at least one anti-FXII antibody (e.g. like F1antibody (MoAb F1, Rayon et al., Blood. 1995 Dec. 1; 86(11):4134-43))and/or the use of at least one protease inhibitor to inhibit FXII-driventhrombus formation is according to the present invention.

Especially preferred is the protease inhibitor selected from for exampleAT III inhibitor, angiotensin converting enzyme inhibitor, Cl inhibitor,aprotinin, alpha-1 protease inhibitor, antipain([(5)-1-Carboxy-2-Phenylethyl]-Carbamoyl-L-Arg-L-Val-Arginal),Z-Pro-Pro-aldehyde-dimethyl acetate, DX88 (Dyax Inc., 300 TechnologySquare, Cambridge, Mass. 02139, USA; cited in: Williams A. and BairdLG., Transfus Apheresis Sci. 2003 Dec. 29 (3):255-8), leupeptin,inhibitors of prolyl oligopeptidase such as Fmoc-Ala-Pyr-CN,corn-trypsin inhibitor, mutants of the bovine pancreatic trypsininhibitor, ecotin, YAP (yellowfin sole anticoagulant protein) andCucurbita maxima trypsin inhibitor-V including Curcurbita maximaisoinhibitors.

Accordingly, the present invention provides the use of such an antibodyand/or inhibitor described herein in medicine; and also the use of suchan antibody and/or inhibitor in the manufacture of a medicament.

Therefore, according to another aspect of the present invention, apharmaceutical formulation is provided comprising at least one antibodyand/or one inhibitor, which is suitable for inhibiting factor XII andwhich prevents the formation and/or the stabilization ofthree-dimensional arterial or venous thrombi.

In particular, the antibody used for the pharmaceutical formulation isan anti-FXII antibody (e.g. like F1 antibody (MoAb F1, Rayon et al.,Blood. 1995 Dec. 1; 86(11):4134-43)), and the inhibitor is a proteaseinhibitor, for example but not limited to AT III inhibitor, angiotensinconverting enzyme inhibitor, C1 inhibitor, aprotinin, alpha-1 proteaseinhibitor, antipain([(S)-1-Carboxy-2-Phenylethyl]-Carbamoyl-L-Arg-L-Val-Arginal),Z-Pro-Pro-aldehyde-dimethyl acetate, DX88 (Dyax Inc., 300 TechnologySquare, Cambridge, Mass. 02139, USA; cited in: Williams A. and BairdLG., Transfus Apheresis Sci. 2003 Dec. 29 (3):255-8), leupeptin,inhibitors of prolyl oligopeptidase such as Fmoc-Ala-Pyr-CN,corn-trypsin inhibitor, mutants of the bovine pancreatic trypsininhibitor, ecotin, YAP (yellowfin sole anticoagulant protein) andCucurbita maxima trypsin inhibitor-V including Curcurbita maximaisoinhibitors.

The antibody may also be a fragment of same or mimetic retaining theinhibitory activity, for example analogues of Kunitz Protease Inhibitordomain of amyloid precursor protein as disclosed in U.S. Pat. No.6,613,890 especially in columns 4 through 8. Other suitable inhibitorsmay be Hamadarin as disclosed by Harahiko Isawa et al. in The Journal ofBiological Chemistry, Vol. 277, No. 31 (August 2, pp. 27651-27658,2002). A suitable Corn Trypsin Inhibitor and methods of its productionare disclosed in Zhi-Yuan Chen et al., Applied and EnvironmentalMicrobiology, March 1999, p. 1320-1324 and reference 19 cited ibidem.All references cited are incorporated for reference including theirentire content in this application. Last not least, small moleculesisolated for example via use of FXII respective FXIIa inhibition as theassay on which selection is based are part of the invention, as well astheir respective use described above or below. These small moleculeFXIIa inhibitors could be designed on the bases of a crystal structureof FXII. Therefore several FXII domains or the light chain could beexpressed recombinantly in expression systems such as E. coli, yeast ormammalian cells. Then the protein is purified and crystallized usingstandard procedures as described for the FXII substrate FXI (Jin L, etal. (2005) Crystal structures of the FXIa catalytic domain in complexwith ecotin mutants reveal substrate-like interactions. J Biol Chem.280(6):4704-12.) Alternatively, small molecule serine proteaseinhibitors could be included to stabilize the FXII structure. Suchformulations comprising small molecule inhibitors of protein targets,which can be for example designed guided by crystals of these targetproteins, are well known in the art and include pharmaceuticalformulations that may be, for example, administered to a patientsystemically, such as parenterally, or orally or topically.

The term “parenteral” as used here includes subcutaneous, intravenous,intramuscular, intra-arterial and intra-tracheal injection,instillation, spray application and infusion techniques. Parenteralformulations are preferably administered intravenously, either in bolusform or as a constant infusion, or subcutaneously, according to knownprocedures. Preferred liquid carriers, which are well known forparenteral use, include sterile water, saline, aqueous dextrose, sugarsolutions, ethanol, glycols and oils.

Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, fillers, lubricants and wettingagents, etc. Oral liquid preparations may be in the form of aqueous oroily suspensions, solutions, emulsions, syrups, elixirs or the like, ormay be presented as a dry product for reconstitution with water or othersuitable vehicle for use. Such liquid preparations may containconventional additives, such as suspending agents, emulsifying agents,non-aqueous vehicles and preservatives.

Formulations suitable for topical application may be in the form ofaqueous or oily suspensions, solutions, emulsions, gels or, preferably,emulsion ointments. Formulations useful for spray application may be inthe form of a sprayable liquid or a dry powder.

According to a third aspect of the present invention, the use of factorXII as an anti-thrombotic target by inhibiting factor XII by at leastone antibody and/or one inhibitor and preventing therefore the formationand/or the stabilization of three-dimensional thrombi in the vessel isprovided.

The nature, benefit, and further features of the present inventionbecome apparent from the following detailed description of the performedexperiments and their results when read in conjunction with theaccompanying figures described below.

Factor XII-deficient mice were used to analyze the function of theintrinsic coagulation cascade in hemostasis and thrombosis. Intravitalfluorescence microscopy and ultrasonic flow measurements revealed asevere defect in the formation and stabilization of three-dimensionalthrombi in different arterial branches of the vascular system.Reconstitution of the mutant mice with human factor XII restored theintrinsic coagulation pathway in vitro and arterial thrombus formationin vivo. Mechanistically, the procoagulant activity of the intrinsicpathway was critically promoted by activated platelets. These resultsplace the FXII-induced intrinsic blood coagulation cascade in a centralposition in the process of arterial thrombus formation linking plasmaticcoagulation with platelet aggregation.

FIGS. 1A, 1B, 1C, and 1D describe the coagulation analysis of FXIIdeficient mice: (A) Tail bleeding times of wild-type (n=12) and FXII−/−(n=11) mice. Each symbol represents one individual. (B) Peripheral bloodcounts in thousands/μl and global coagulation parameters of FXII−/− andwt mice. The abbreviations are white blood counts (WBC), activatedpartial thromboplastin time (aPTT) and prothrombin time (PT). Valuesgive mean values±SD of 10 mice of each genotype. (C) Contact systemproteins FXII, plasma kallikrein (PK) and high molecular weightkininogen (HK) probed in 0.3 μl wt and FXII−/− plasma by Westernblotting using specific antibodies. A molecular weight standard is givenon the left. (D) Recalcification clotting times were determined inplatelet free (upper panel) and platelet rich (lower panel) plasma fromC57BL/6 and 129sv wt, FXII−/−, FcRγ−/− and integrin α2-deficient micefollowing activation with kaolin (dark columns) or collagen (lightcolumns). The effect of JON/A was analyzed in C57BL/6 plasmasupplemented with 50 μg/ml antibody. Means±STD from 6 experiments aregiven.

FIG. 2(A) Thromboembolic mortality was observed following theintravenous injection of collagen (0.8 mg/kg) and epinephrine (60μg/kg). All wild-type mice died within 5 min. Animals that were alive 30min after challenge were considered survivors. FIG. 2(B) Platelet countsin control (n=19), FXII−/− (n=14) and FcRγ−/− (n=5) mice 2 min afterinfusion of collagen/epinephrine. FIG. 2(C) Heparinized platelet richplasma from wild-type and FXII−/− mice was stimulated with collagen (10μg/m1) or ADP (5 μM) and light transmission was recorded in a standardaggregometer. The results shown are representative of six mice pergroup. FIG. 2(D)

Hematoxilin/Eosin-stained sections from lungs of the indicated mice 2min after collagen/epinephrine injection. Thrombi per eyefield warecounted in 20× magnification. The bars represent means ±SDT from 100eyefields.

FIGS. 3A, 3B, 3C, and 3D describe the defective thrombus formation inmice lacking factor XII in vivo. Thrombus formation in vivo wasmonitored on mesenteric arterioles upon injury induced with 20% FeCl3.(A) Single platelet adhesion is detected 5 min after injury in all mousestrains, 7 to 8 minutes after injury the first thrombi in wt mice wereobserved, whereas in FXII−/− the first thrombi occurred 14 to 35 minutesafter injury and in FXI−/− 5 to 35 minutes after injury. (B) Thrombusformation was observed in 100% of mesenteric arteries in wild type mice,but only in 50% of FXII−/− mice and in 44.4% of FXI−/− mice. (C) Thrombiformed in wt mice occluded the vessel in average 25 minutes after injurywhereas thrombi formed in FXII and FXI deficient mice did not lead toocclusion. Each symbol represents one single monitored arteriole. (D)Representative pictures of one experiment.

FIG. 4(A) Wild-type (n=10), FXII−/− (n=10) and FXI−/− (n=11) wereanalyzed in an arterial occlusion model. Thrombosis was induced in theaorta by one firm compression with a forceps. Blood flow was monitoredwith an perivascular ultrasonic flow probe until complete occlusion. Theexperiment was stopped after 40 min. Each symbol represents oneindividual. FIG. 4(B) Mechanical injury in the carotid artery wasinduced by a ligation. After removal of the filament thrombus area inwild-type (n=10) and FXII−/− (n=10) was measured in μm2. FIG. 4(C) Thephotomicrographs show representative images 2 min after injury.

FIGS. 5A, 5B, 5C, and 5D describe the defect in thrombus formation inFXII deficient animals which is restored by human FXII. (A) Thrombusformation upon FeCl₃ induced injury was observed in 100% of mesentericarteries in wild-type mice as well as in FXII−/− mice injected withhuman FXII. (B) Formed thrombi occluded the vessel in average 25 minutesafter injury in wild-type mice and in 22.7 minutes after injury inFXII−/− mice injected with human FXII. Each symbol represents oneindividual. (C) Representative pictures are shown. (D) FXII−/− micereceived 2 mg/kg hFXII−/− and thrombosis was induced in the aorta by onefirm compression with a forceps. Blood flow was monitored with anperivascular ultrasonic flow probe until complete occlusion. Theexperiment was stopped after 40 min. Each symbol represents oneindividual.

FIGS. 6A, 6B, and 6C describe the anti-FXII antibodies inhibitingthrombus formation in mice in vivo. Wild-type mice received 2 mg/kganti-FXII antibodies or non-immune IgG i.v. After 15 min, thrombusformation in vivo was monitored on mesenteric arterioles upon injuryinduced with 20% FeCl₃. (A) Single platelet adhesion is detected 5 minafter injury in both groups. After 7 to 8 minutes the first thrombi micein control IgG-treated mice were observed, whereas in anti-FXII-treatedmice the first thrombi occurred 12 to 32 minutes after injury. (B)Thrombus formation was observed in 100% of mesenteric arteries incontrol mice, but only in 60% of anti-FXII-treated mice. (C) Time tocomplete occlusion is shown. Each symbol represents one individual.

FIGS. 7A and 7B describe a revised model of arterial thrombus formation.(A) Initially, at sites of vascular lesions thrombin formation ispredominantly due to tissue factor (TF) exposure in the subendothelialmatrix. TF in complex with FVII initiates the extrinsic pathway ofcoagulation. At the site of injury the contribution of the FXII drivingthe intrinsic pathway via FXI for thrombin (FII) generation is minor andnegligible for normal hemostasis. Accordingly individuals withFXII-deficiency do not suffer from bleeding. Generated thrombininitiates clot formation by forming fibrin and activating platelets. (B)Propagation of thrombus growth: On surfaces exposed in the growingthrombus the FXII-induced intrinsic pathway critically contributes tothrombin generation. Activated FXII generates additional fibrin throughFXI. Accordingly, FXII- as well as FXI-deficiency severely impairsthrombus formation.

In the present invention a potential contribution of the intrinsicpathway of coagulation for pathological thrombus formation in vivo wasassessed by intravital microscopy- and flow measurement-based models ofarterial thrombosis using mice lacking factor XII. While initialadhesion of platelets at sites of injury is not altered in the mutantanimals, the subsequent formation and stabilization of three-dimensionalthrombi is severely defective. This defect was seen in differentbranches of the vasculature and could be completely restored byexogenous human factor XII. These findings establish the factorXII-driven intrinsic coagulation pathway as a major link between primaryand secondary hemostasis in a revised model of thrombus formation.

To analyze the function of FXII for clotting in vivo, FXII-deficientmice were generated. FXII−/− mice are healthy, phenotypicallyindistinguishable from their wild-type littermates, and fertile.Detailed histological and hemostasiological analyses showed nocorrelates for increased thrombosis or bleeding in FXII−/− mice despitea prolonged aPTT of 68±17 sec and recalcification time of 412±78 sec inretroorbitally collected plasma (wt: 23±4 and 210±31 sec) (Pauer, H. U.,et al. (2004). Targeted deletion of murine coagulation factor XII gene-amodel for contact phase activation in vivo. Thromb. Haemost. 92,503-508). Similarly to FXII-deficient humans, FXII−/− mice present withno increased bleeding tendency as indicated by tail bleeding timessimilar to those found in wild-type animals (369.5±201.7 and 355.9±176.1sec, respectively, n=12 per group, FIG. 1A). Peripheral blood cellcounts of mutant mice did not differ from wild-type controls. Notably,the prothrombin time (PT) of FXII−/− mice was similar to the wild-type(8.9±1.3 vs. 9.1±1.3 sec) indicating that FXII deficiency does notaffect fibrin formation by the extrinsic coagulation system (FIG. 1B).To assess potential differences in FXII procoagulant activity betweenhumans and mice, FXII-deficient human (FXII<1%) with murine wild-typeplasma or vice versa were reconstituted and the PTT of the mixtures wasdetermined. In either case, clot formation was normalized supporting thenotion that FXII function for clotting is comparable in humans and mice.

In humans similarly to FXII deficiency the deficiency of contact systemproteins plasma kallikrein (PK) and high molecular weight kininogen (HK)does not result in an increased bleeding risk despite a prolonged aPTT.To confirm that the aPTT prolongation in FXII−/− mice is not due toadditional defects of contact phase proteins, we analyzed PK and HK inFXII−/− and wt plasma. The Western blot indicated that HK and PK levelsare equivalent in mutant and wild-type mice (FIG. 1C). Functionally, inFXII−/− plasma exposed to collagen or kaolin HK procession and thrombinformation was severely impaired compared to wild-type.

Blood coagulation and platelet activation are complementary and mutuallydependent processes. Platelets interact with and contribute to theactivation of several coagulation factors and the central coagulationproduct thrombin is a potent platelet activator. Therefore, next thecontribution of platelets and FXII was examined to clot formation inmore detail. For this, we induced clotting using either kaolin thatclassically activates FXII but has no direct effect on platelets orcollagen, which activates both FXII and platelets where it interactswith numerous receptors, most importantly α2β1 integrin and GPVI. In thepresence, but not in the absence of platelets, collagen was superior tokaolin for clot formation in wild-type plasma (FIG. 1D). In contrast, inplasma containing activation-defective FcRγ−/− platelets, the relativepotency of kaolin and collagen was similar to PFP and a similar effectwas seen with PRP from integrin α2−/− mice. Platelet procoagulantactivity is also efficiently triggered in coagulating plasma and thefibrin(ogen) receptor αIIbβ3 has been shown to play a crucial role inthis process although the underlying mechanisms are not fullyunderstood. In agreement with these reports, the αIIbβ3-functionblocking antibody JON/A largely inhibited the platelet-dependentdecrease in the clotting time (FIG. 1D). Together, these resultsdemonstrated that platelets in a procoagulant state can promoteFXII-induced clot formation.

To test whether collagen-induced FXII activation has functionalconsequences in vivo, wild-type and FXII−/− mice were subjected to amodel of lethal pulmonary thromboembolism induced by the infusion of amixture of collagen (0.8 mg/kg body weight) and epinephrine (60 μg/kgbody weight). All of the control mice (19/19) died within 5 min fromwidespread pulmonary thrombosis and cardiac arrest which was accompaniedby a >95% reduction in circulating platelet counts as soon as 2 minafter challenge (FIG. 2A, B). Under these experimental conditions, 35.7%(5/14) of the FXII−/− mice survived although their peripheral plateletcounts were similarly reduced as in the wild-type control, suggestingthat the observed protection was not based on a platelet activationdefect. This assumption was confirmed by in vitro studies showing thatFXII−/− platelets express normal levels of the major surfaceglycoproteins, including collagen receptors, and that the cells arenormally activatable by classical agonists such as thrombin, adenosinediphosphate (ADP), or the GPVI-specific agonist, collagen-relatedpeptide (as measured by activation of integrin αIIbβ3 and P-selectinexpression). In agreement with this, FXII−/− platelets exhibited anunaltered aggregation response to collagen, ADP (FIG. 2C), PMA, orthrombin.

In a parallel set of experiments, FcRγ−/− mice were challenged withcollagen/epinephrine. These mice were completely protected fromlethality and platelet counts were only moderately reduced 2 min afterchallenge confirming the strict requirement for platelet activation forlethality in this model. These data were further supported byhistological analysis of lung sections derived from mice of thedifferent groups. While the large majority of the vessels was obstructedin wild-type mice, this was significantly reduced in FXII−/− mice(survivors and non survivors). In agreement with previous reports,virtually no thrombi were found in lungs from FcRγ−/− mice (FIG. 2D).These results suggested that in vivo collagen triggers both plateletactivation and the FXII-dependent intrinsic coagulation pathway which inthis model synergize to form occlusive pulmonary thrombi.

Pathological thrombus formation is frequently initiated by fissuring orabrupt disruption of the atherosclerotic plaque in the arterial branchof the vasculature leading to unphysiologically strong plateletactivation and procoagulant activity on the surface on thesubendothelial layers. To test the role of FXII in these processes,thrombus formation was studied in wild-type and FXII−/− mice, employingdifferent models of arterial injury. In the first model, oxidativeinjury was induced in mesenteric arterioles (60-100 μm in diameter) andthrombus formation was examined by in vivo fluorescence microscopy.Wild-type and FXII−/− mice received fluorescently labeled platelets(1×108) of the same genotype and injury was induced by topicalapplication of a filter paper saturated with 20% ferric chloride (FeCl₃)for 1 min which provokes the formation of free radicals leading to thedisruption of the endothelium. Platelet interactions with the injuredvessel wall started rapidly and five minutes after injury the number offirmly adherent platelets was similar in both groups of mice (FIG. 3A).However, while in wild-type mice the adherent platelets consistentlyrecruited additional platelets from the circulation, resulting in theformation of aggregates, this process was severely defective in themutant mice. In 100% of the control vessels (17/17), stable thrombi >20μm in diameter had formed with 10 min after injury which continuouslygrew over time and finally lead to complete occlusion in 94.1% (16/17)of the vessels within the observation period of 40 min (mean occlusiontime: 25.6±8.9 min)(FIG. 3). In sharp contrast, in mutant mice theformation of microaggregates or thrombi occurred was completely absentin 50% (7/14) of the vessels. In the remaining 50% (7/14) of thevessels, thrombi were formed which were, however, consistently unstableand rapidly detached from the vessel wall. In none of the vessels, athrombus >20 μm in diameter remained attached at the site of injury formore than 1 min. Consequently, no vessel occluded in FXII−/− mice withinthe observation period (40 min). This unexpected result demonstratedthat FXII is required for the generation and stabilization ofplateletrich thrombi in FeCl₃-injured arterioles and suggested thatFXII-induced the coagulation pathway essentially contributes to theobserved thrombotic response. This assumption was confirmed when micedeficient in FXI were analysed in the same model. Since FXI is theprincipal substrate of FXII in the “intrinsic” cascade, a similar defectin thrombus formation would have to be expected in those mice. Indeed,very similar to FXII−/− mice, virtually normal platelet adhesion at thesite of injury was detectable during the first three minutes afterinjury, whereas the formation of thrombi was completely inhibited in55.6% (5/9) of the vessels. In the remaining vessels, the formedmicroaggregates and thrombi were unstable and continuously embolized(FIG. 3). As a result, none of the vessels occluded within theobservation period (40 min). This data shows that FXI-deficient mice areprotected in a model of FeCl₃-induced occlusion of the carotid artery.

FeCl₃-induced arterial thrombus formation is known to depend onplatelets and thrombin generation but it is unclear how well this typeof injury resembles the thrombogenic milieu produced in diseasedvessels, e.g. upon rupture of the atherosclerotic plaque. Therefore, toexclude the possibility that the massive FeCl₃-induced oxidative damageproduces unphysiological conditions which may artificially favorFXII-dependent contact phase activation, the function of FXII wasassessed in a second well-established arterial thrombosis model whereinjury is induced mechanically in the aorta and blood flow is monitoredwith an ultrasonic flow probe. After a transient increase directly afterinjury, blood flow progressively decreased for several minutes in allmice tested. In all tested wild-type mice (10/10), this decreaseresulted in complete and irreversible occlusion of the vessel within 1.6to 11.1 min after injury (mean occlusion time 5.3±3.0 min, FIG. 4A). Adifferent picture was found in FXII−/− mice where stable thrombusformation was severely defective. While in all animals a progressivereduction in blood flow was observed during the first minutes afterinjury, occlusion occurred only in 4 of 10 mice. Moreover, the occlusivethrombi in those mice were in all cases unstable and rapidly embolizedso that blood flow was re-established between 10 s and 115 s afterocclusion. None of the re-opened vessels occluded a second time.Consequently, all FXII−/− mice displayed essentially normal flow ratesthrough the injured vessel at the end of the observation period (40min). Very similar results were obtained with FXI−/− mice, where 9 of 11were unable to establish an occlusive thrombus within the observationperiod (30 min)(FIG. 5A).

The severe defect in arterial thrombus formation in FXII−/− mice wasconfirmed in a third independent model where platelet recruitment in theinjured carotid artery was studied by in vivo fluorescence microscopy.Platelets were purified from donor mice, fluorescently labeled andinjected into recipient mice of the same genotype. Vascular injury wasinduced by vigorous ligation of the carotid artery which consistentlycauses disruption of the endothelial layer and frequently breaching ofthe internal elastic lamina followed by rapid collagen-triggeredplatelet adhesion and thrombus formation at the site of injury (Gruneret al., Blood 102: 4021-27, 2003). While wild-type animals rapidlyformed large stable thrombi (thrombus area: 102.821±39.344 μm2; t=5min), which did not embolize, only small and medium-sized aggregatesformed in the mutant mice, which were frequently detached from the siteof injury (FIG. 4B, C). Consequently, the thrombus area was dramaticallyreduced in the mutant mice (8.120±13.900 μm2; t=5 min) although primaryplatelet adhesion on the vessel wall appeared not to be defective. Totest whether the severe defect in thrombus formation in FXII−/− miceresults from the lack of plasma FXII or platelet FXII, or possibly fromsecondary, unidentified effects of FXII deficiency such as alterationsin the vasculature, arterial thrombus formation was studied in FXII−/−mice following administration human of FXII (2 μg/g body weight). Thistreatment normalized the PTT (27±6 sec) and fully restored arterialthrombus formation. In 100% of the FeCl3-injured mesenterial arterioles,thrombi >20 μm had formed within 10 min after injury and all of thevessels completely occluded within the observation period (FIG. 5A-C).There was even a tendency towards faster occlusion detectable in thereconstituted FXII−/− mice as compared to untreated wild-type controlmice (mean occlusion time: 22.7±8.2 min vs. 25.6±8.9 min). A similarresult was obtained when injury was induced mechanically in the aorta.In all tested vessels, complete and irreversible occlusion occurredwithin 10 min after injury (FIG. 5D), confirming that the lack of plasmaFXII accounts for the thrombotic defect observed in FXII−/− mice.

The above-described studies demonstrated that FXII is crucial forarterial thrombus formation and may, therefore, serve as anantithrombotic target.

To assess this directly, mice were treated with 2 mg/kg body weightpolyclonal rabbit anti-mouse FXII antibodies or non-immune rabbitantibodies and assessed platelet recruitment and thrombus formation inmesenterial arteries following FeCl₃-induced injury. As shown in FIG.6A, platelet adhesion at sites of injury was comparable in both groupsof mice. However, while in 100% of the control vessels, thrombi >20 μmhad formed within 10 min after injury and all of the vessels completelyoccluded within the observation period (FIG. 6B, C), thrombi >20 μm wereonly observed in 67% of the vessels and occlusion occurred only in 50%of the vessels of the animals treated with anti-FXII antibody.

Alternatively, to test the impact of small molecule FXII inhibitors,wildtype mice were infused with the FXII inhibitor corn trypsininhibitor (CTI, 50 μg/g body weight) 5 min prior to FeCl₃-induced injuryin the carotic artery (Wang et al. (2005) J. Thromb. Heamost. 3:695-702). Inhibitor treatment prolonged the aPTT (62±11 sec, n=4) butdid not affect bleeding during the surgical procedure. In none of theanimals tested (0/4) vessel occluding thrombi developed within 30 minfollowing application of FeCl₃.

These results demonstrated that anti-FXII therapeutics like anti-FXIIantibodies or small molecule FXII inhibitors provide significantprotection against arterial thrombus formation.

Although contact activation of FXII has been recognized as the startingpoint of the intrinsic blood coagulation cascade for more than 50 yearsthis pathway was considered to be irrelevant for blood clotting. In thepresent invention, three different in vivo models were used to analyzeplatelet recruitment and thrombus formation at sites of arterial injuryin FXII-deficient mice by in situ video microscopy and ultra-sonic flowmeasurements and showed a severe inability to form stablethree-dimensional thrombi. This defect was based on the lack of FXII inplasma, but not other compartments, as it was completely reversed byintravenous injection of exogenous human FXII (FIG. 6) thereby alsoexcluding that secondary effects of FXII deficiency contribute to theobserved phenotype.

These results are unexpected as FXII has been regarded as anantithrombotic rather than a prothrombotic enzyme based on a few reportsshowing an association of FXII-deficiency with an increased incidence ofvenous thrombosis (Kuhli, C., Scharrer, I., Koch, F., Ohrloff, C., andHattenbach, L. O. (2004). Factor XII deficiency: a thrombophilic riskfactor for retinal vein occlusion. Am. J. Ophthalmol. 137, 459-464;Halbmayer, W. M., Mannhalter, C., Feichtinger, C., Rubi, K., andFischer, M. (1993). [Factor XII (Hageman factor) deficiency: a riskfactor for development of thromboembolism. Incidence of factor XIIdeficiency in patients after recurrent venous or arterialthromboembolism and myocardial infarction]. Wien. Med. Wochenschr. 143,43-50). FXII-deficient mice display normal bleeding times (FIG. 1A) anddo not show signs of spontaneous or increased posttraumatic(intraoperative) bleeding confirming that FXII is dispensable for normalhemostasis. At the first sight, these results seem to contradict with acentral dogma of hemostasis that only those factors whose deficiency isassociated with bleeding or thrombosis are relevant to blood clotting.On a closer look, however, the data do not challenge this theorem butrather raise the interesting possibility that hemostasis and arterialthrombosis may occur through different mechanism.

Although the above discussed mechanisms of sustained thrombin generationmay be sufficient to generate a hemostatic plug, the data show that theformation of stable arterial thrombi requires the additional activationof the intrinsic coagulation pathway, at least in mice. There is noevidence for the possibility that species-specific differences exist inthe function of FXII or a substrate of the enzyme. All coagulationparameters and the hemostatic phenotype of the mutant mice are in linewith human FXII-deficiency and all alterations observed in animals werenormalized by reconstitution with human FXII (FIG. 5). Furthermore, itis excluded that the thrombotic defect is restricted to a particularexperimental model as it was found in different arterial branches of thevasculature and independent of the type of injury. It may be difficultto determine what type of damage best reflects the vascular lesionproduced by rupture of an atherosclerotic plaque, which is consideredthe major trigger of acute cardiovascular syndromes. Atheroscleroticlesions are rich in thrombogenic constituents, most importantly TF andfibrillar collagens. In the process of atherogenesis, enhanced collagensynthesis by intimal smooth muscle cells and fibroblasts has been shownto significantly contribute luminal narrowing. Plaque rupture orfissuring results in exposure of collagen fibrils to the flowing bloodwhich triggers platelet adhesion and aggregation. In addition, theyinduce FXII activation as shown here for fibrillar collagen type I,which is the major collagen type found in the vessel wall. But thecollagens are likely not the only (patho)physiological activator of FXIIat sites of injury. Other candidates could be substances liberated fromdisintegrating cells or exposed in the ECM including HSP90 or solubleand insoluble polyanions, e.g. nucleosomes or glycosaminogly-cans.

Among these FXII activators, collagens are by far most thrombogenicbecause they also potently activate platelets. At sites of injury,platelets tether to the ECM by the reversible interaction of plateletGPIb-V-IX with collagen-bound vWf which reduces the velocity of thecells and thereby allows binding of other receptors. Among these, thecollagen receptor GPVI is of central importance as it activatesintegrins α2β1 and αIIbβ3 which then mediate stable adhesion andcontribute to cellular activation. In addition, platelet activationthrough the GPVI/FcRγ-chain complex induces a procoagulant state of thecells which is characterized by the exposure of phosphati-dylserine (PS)and the production of (PS exposing) membrane blebs and microve-sicles.Integrin α2β1 facilitates this process directly by “outside-in” signalsand indirectly by reinforcing GPVI-collagen interactions. It isestablished that PS-containing membranes strongly accelerate two centralreactions of the coagulation process, the tenase and prothrombinasereactions. The present invention shows that procoagulant plateletsfacilitate FXII-dependent clotting in vitro by a mechanism involvingboth the GPVI/FcRγ-chain complex as well as α2β1 (FIG. 2). This could atleast partly explain why α2β1-deficient mice, despite unaltered plateletadhesion at sites of arterial injury, show partial defects in theformation of occlusive thrombi. Besides collagens, coagulating plasmapotently stimulates platelet procoagulant activity by an integrinαIIbβ3-dependent mechanism. In the present experiments, αIIbβ3 blockadealmost completely inhibited platelet participation in FXII-dependentclotting, indicating that the well-known anticoagulant activity ofαIIbβ3 antagonists may partly be based on the inhibition of theFXII-driven intrinsic coagulation pathway. Together, the presentinvention indicates that the FXII-driven contact system and plateletactivation may be mutually dependent processes that cooperate inpathological thrombus formation.

Based on the experimental results, a model of pathological thrombusformation depicted schematically in FIG. 7 was proposed. At sites ofvascular injury, the first layer of platelets comes in contact withcollagens in an environment that is additionally enriched in TF andfibrin. It is therefore not surprising that platelet adhesion to thedamaged vessel wall is not impaired in FXII−/− mice and it is verylikely that these cells were fully activated and in a procoagulantstate. In a growing thrombus, however, collagens are absent and TFconcentrations provided by microvesicles may be lower as compared to thevessel wall and reduced in their activity by TFPI released in largeamounts from activated platelets. Under these conditions, additionalmechanisms are required to maintain spatio-temporal thrombin generationto activate newly recruited platelets and, via the formation of fibrinprovoke their coagulant activity. The severe inability of FXII−/− miceto establish stable thrombi unambiguously demonstrates that theFXII-driven intrinsic coagulation pathway is an essential player in thisprocess. Together with the observation that low TF-mice also displayimpaired arterial thrombosis, these results suggest that both extrinsicand intrinsic pathway must be operative and synergize to promote theformation of a three-dimensional and eventually occluding thrombus. Incontrast, the lack of bleeding in FXII−/− mice indicates that thrombusgrowth in the third dimension may not be necessary to seal a hole in thevessel wall. This could explain why the extrinsic pathway, whichproduces the first thin layer of fibrin and activated platelets, issufficient to mediate normal hemostasis. Our results raise theinteresting possibility the formation of a three-dimensional thrombusserves functions other than hemostasis. These could include the arrestof blood flow in certain areas of tissue trauma in order to prevent thedistribution of invading pathogens or toxins with the blood stream.

Experimental Procedures Animals

All experiments and care were approved by the local Animal Care & UseCommittee. Classical mouse mutants lacking factor XI (FXI−/−), factorXII (FXII−/−), α2 integrin (α2−/−) were produced as described (Gailani,D., Lasky, N. M., and Broze, G. J., Jr. (1997). A murine model of factorXI deficiency. Blood Coagul. Fibrinolysis 8, 134-144; Pauer, H. U.,Renne, T., Hemmerlein, B., Legler, T., Fritzlar, S., Adham, I.,Muller-Esterl, W., Emons, G., Sancken, U., Engel, W., and Burfeind, P.(2004). Targeted deletion of murine coagulation factor XII gene-a modelfor contact phase activation in vivo. Thromb. Haemost. 92, 503-508;Holtkotter, O., Nieswandt, B., Smyth, N., Muller, W., Hafner, M.,Schulte, V., Krieg, T., and Eckes, B. (2002). Integrin alpha 2-deficientmice develop normally, are fertile, but display partially defectiveplatelet interaction with collagen. J Biol Chem JID—2985121R 277,10789-10794). As a control C57B/6J mice (FXI−/−) or Sv129 (FXII−/−) wereused. Mice deficient in the FcRγ-chain (Takai, T., Li, M., Sylvestre,D., Clynes, R., and Ravetch, J. V. (1994). FcR gamma chain deletionresults in pleiotrophic effector cell defects. Cell 76, 519-529) werefrom (Taconics, Germantown).

Generation of Anti-FXII Antibodies

Total cellular RNA was isolated from a liver of a 129sv wt mouse and theFXII-cDNA synthesis was performed with the “one-step RT-PCR Kit” fromQiagen according to the manufacturers instructions. The factor FXIIheavy chain (positions 61-1062, corresponding to residues 21-354) wasamplified using 25 pmol each of the 5- and 3-primers(ttggatccccaccatggaaagactccaag and ttgaattcgcgcatgaacgaggaca g)introducing a BamH I and EcoR I restriction site, respectively with thefollowing protocol: 30 s at 95° C., 60 s at 58° C., and 1 min at 72° C.for 30 cycles on a thermal cycler (Biometra, Göttingen, Germany). ThePCR product was cloned into the BamH I and EcoR I site of the pGEX-2Texpression vector (Pharmacia). Following sequencing protein wasexpressed in E.coli strain BL21. Exponentially growing bacteria werestimulated with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 1 h,harvested, resuspended in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA,200 mM NaCl, 10 μg/ml benzamidine hydrochloride, 10 μg/mlphenylmethylsulfonyl fluoride and sonicated for 3 min in pulses of 15 seach. After centrifugation at 15,000×g for 20 min at 4° C., thesupernatant was removed and transferred to a GST-sepharose column(Pharmacia) for purification. Eluted protein was >95% pure as deducedfrom Coomassie stained SDS-PAGE. Polyclonal antibodies against GST-heavychain FXII were raised in rabbits following standard procedures.Antibodies were selected from the hyperimmunserum using columns withFXII-heavy chain fused to the maltose binding protein (MBP). Thesefusion proteins were expressed and purified using the pMAL-c2 expressionsystem and amylose resin columns similarly as described for theGST-fusion construct.

Platelet Preparation

Mice were bled under ether anesthesia from the retroorbital plexus.Blood was collected in a tube containing 20 U/mL heparin, and plateletrich plasma (prp) was obtained by centrifugation at 300 g for 10 min atroom temperature (RT). For washed platelets, prp was centrifuged at 1000g for 8 min and the pellet was resuspended twice in modifiedTyrodes-Hepes buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mMNaHCO3, 20 mM Hepes, 5 mM glucose, 0.35% bovine serum albumin, pH 6.6)in the presence of prostacyclin (0.1 μg/ml) and apyrase (0.02 U/mL).Platelets were then resuspended in the same buffer (pH 7.0, 0.02 U/mL ofapyrase) and incubated at 37° C. for at least 30 min before analysis.

Flow Cytometry

Heparinized whole blood was diluted 1:20 with modified Tyrode-HEPESbuffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mMHEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], pH 7.0)containing 5 mM glucose, 0.35% bovine serum albumin (BSA), and 1 mMCaCl2. The samples were incubated with fluorophore-labeled antibodiesfor 15 minutes at room temperature and directly analyzed on aFACScalibur (Becton Dickinson, Heidelberg, Germany) (Nieswandt, B.,Schulte, V., and Bergmeier, W. (2004). Flow-cytometric analysis of mouseplatelet function. Methods Mol. Biol. 272, 255-268).

Aggregometry

To determine platelet aggregation, light transmission was measured usingprp (200 μL with 0.5×106 platelets/μL). Transmission was recorded in aFibrintimer 4 channel aggregometer (APACT Laborgerate andAnalysensysteme, Hamburg, Germany) over 10 min and was expressed asarbitrary units with 100% transmission adjusted with plasma. Plateletaggregation was induced by addition of collagen (10 μg/mL) and ADP (5μM).

Bleeding Time Experiments

Mice were anesthetized by intraperitoneal injection of tribromoethanol(Aldrich) (0.15 m1/10 g of body weight) and 3 mm segment of the tail tipwas cut off with a scalpel. Tail bleeding was monitored by gentlyabsorbing the bead of blood with a filter paper without contacting thewound site. When no blood was observed on the paper after 15 secondintervals, bleeding was determined to have ceased. When necessary,bleeding was stopped manually after 20 minutes. Where indicated, micewere treated with 100 μg/mouse of hFXII.

Preparation of Platelets for Intravital Microscopy

Mouse blood (1 vol) was collected into 0.5 vol of Hepes buffercontaining 20 U/mL heparin. The blood was centrifuged at 250 g for 10minutes and platelet-rich plasma was gently transferred to a fresh tube.Platelets were labelled with 5-carboxyfluorescein diacetate succinimidylester (DCF) and adjusted to a final concentration of 200×106platelets/250 μl (Massberg, S., Sausbier, M., Klatt, P., Bauer, M.,Pfeifer, A., Siess, W., Fassler, R., Ruth, P., Krombach, F., andHofmann, F. (1999). Increased adhesion and aggregation of plateletslacking cyclic guanosine 3′,5′-monophosphate kinase I. J Exp Med 189,1255-1264).

In Vivo Thrombosis Model with FeCl3-Induced Injury.

Male and female mice in the age of 4-5 weeks were anesthetized byintraperitoneal injection of 2,2,2-tribromoethanol and2-methyl-2-butanol (Sigma) (0.15 m1/10 g of body weight from 2.5%solution). Fluorescently labeled platelets were injected intravenously.Mesentery was exteriorized gently through a midline abdominal incision.Arterioles (35-50 μm diameter) were visualized with a Zeiss Axiovert 200inverted microscope (x10) equipped with a 100-W HBO fluorescent lampsource and a CCD camera (CV-M300) connected to an S-VHS video recorder(AG-7355, Panasonic, Matsushita Electric, Japan). After topicalapplication of FeCl3 (20%) which induced vessel injury and denudation ofthe endothelium, were arterioles monitored for 40 min or until completeocclusion occurred (blood flow stopped for >1 min). Firm plateletadhesion is determined as number of fluorescently labeled platelets thatdeposited on the vessel wall until 5 minutes after injury, thrombus ischaracterized as a platelet aggregate in a diameter larger than 20 μm,occlusion time of vessel is characterized as time required for blood tostop flowing for at least one minute. In all experiments maximum of twoarterioles were chosen from each mouse based on quality of exposure. Atotal of 17 wt, 14 FXII−/− and 9 FXI−/− arterioles were studied.

Intravital Microscopy—Carotid Artery

Intravital microscopy of the injured carotid artery was performedessentially as described (Massberg, S., Gawaz, M., Gruner, S., Schulte,V., Konrad, I., Zohlnhofer, D., Heinzmann, U., and Nieswandt, B. (2003).A crucial role of glycoprotein VI for platelet recruitment to theinjured arterial wall in vivo. J Exp Med JID—2985109R 197, 41-49).Briefly, mice were anesthetized by intraperitoneal injection ofketa-mine/xylazine (ketamine 100 mg/kg, Parke-Davis, Karlsruhe, Germany;xylazine 5 mg/kg, Bayer AG, Leverkusen, Germany). Polyethylene catheters(Portex, Hythe, England) were implanted into the right jugular vein andfluorescent platelets (200×106/250 μl) were infused intravenously.Carotid injury for endothelial denudation was induced by vigorousligation. Prior to and following vascular injury, the fluorescentplatelets were visualized in situ by in vivo video microscopy of theright common carotid artery using a Zeiss Axiotech microscope (20×waterimmersion objective, W 20×/0.5, Zeiss, Göttingen, Germany) with a 100 WHBO mercury lamp for epi-illumination. Platelet adhesion and thrombusformation was recorded for 5 min after the induction of injury and thevideo-taped images were evaluated using a computer-assisted imageanalysis program (Visitron, Munich, Germany).

Pulmonary Thromboembolism

Mice were anesthetized by intraperitoneal injection of2,2,2-tribromoethanol and 2-methyl-2-butanol (Aldrich) (0.15 ml/10 g ofbody weight from 2.5% solution). Anesthetized mice received a mixture ofcollagen (0.8 mg/kg) and epinephrine (60 μg/kg) injected into thejugular vein. The incisions of surviving mice were stitched, and theywere allowed to recover. Necroscopy and histological studies wereperformed on lungs fixed in 4% formaldehyde and paraffin sections werestained with hematoxylin/eosin.

Platelet Count

Platelet count was determined by flow cytometry on a FACScalibur (BectonDickinson, Heidelberg, Germany). Results are expressed as mean±S.D or aspercent of control (wt, n=19; FXII−/−, n=14 and FcRγ−/−, n=5).

Occlusion Time

The abdominal cavity of anesthetized mice was longitudinally opened andthe abdominal aorta was prepared. An ultrasonic flow probe was placedaround the aorta and thrombosis was induced by one firm compression witha forceps. Blood flow was monitored until complete occlusion occurred.The experiment was stopped manually after 45 minutes. Where indicated,human Factor XII was substituted intravenously directly before theexperiment.

Histopathologic Analyses

Mice were sacrificed, lungs rapidly removed and fixed at 4 C for 24 hrin buffered 4% formalin (pH 7.4; Kebo). Tissues were dehydrated andimbedded in paraffin (Histolab Products AB), cut into 4 μm sections, andmounted. After removal of the paraffin, tissues were stained with Mayershematoxylin (Histolab Products AB) and eosin (Surgipath MedicalIndustries, Inc.).

SDS-Polyacrylamide Gel Electrophoresis, Western Blotting, andImmunoprinting

Plasma (0.3 μl/lane) was separated by 12.5% (w/v) polyacrylamide gelelectrophoresis in the presence of 1% (w/v) SDS (Laemmli, 1970).Proteins were transferred onto nitrocellulose membranes for 30 min at100 mA. The membranes were blocked with PBS containing 4% (w/v) dry milkpowder and 0.05% (w/v) Tween-20, pH 7.4. Membranes were probed with 0.5μg/ml of the monoclonal antibody against MBK3 (Haaseman J. Immunology1988). Bound antibodies were detected using peroxidase-conjugatedsecondary antibodies against mouse IgG (dilution 1:5000) followed by achemiluminescence detection method.

Coagulation Assays.

For the determination of the recalcification clotting time, 100 μlcitrate anticoagulated mouse plasma (0.38% sodium citrate), wasincubated with 100 μl each of Horm type collagen (Nycomed, München,Germany), ellagic acid, chondroitin sulfate (both from Sigma), kaolin orbuffer (final concentrations 30 μg/ml) for 120 sec at 37° C. in a KC10“Kugelkoagulometer” (Amelung, Lemgo, Germany). To test the effect ofplatelets activation on FXII-dependent clotting washed platelets wereresuspended in Tyrode buffer including 4 mM Ca2+ and 5 μM Ca2+-ionophorA23187 (Sigma) for 10 min prior to addition to platelet free plasma.Clot formation was initiated by recalcification with 100 μl 25 mMCaCl2-solution, and the time until clotting occurred was recorded usingthe coagulation timer KC4 (Amelung).

Coagulation Analysis

Global and single coagulation parameters were determined with anautomated blood coagulation system (BCS, Dade Behring) with Dade Behringreagents according to the protocols for human samples detailed by themanufacturer. Principles of BCS assay protocols are available from DadeBehring package inserts, which can be found on Dade Behring's web site(http://www.dadebehring.com). D-dimers werde measured with the ELISAfrom Asserachrom (Roche). Peripheral blood counts were determines on theSysmex XE 2100 according to standard protocols.

Thrombin Measurements

Thrombin generation was measured according to the method of Aronson etal. (Circulation, 1985), with slight modifications. Platelet-rich orplatelet free plasma aliquots (0.5 ml) were placed into round-bottomedpolypropylene tubes that were coated with Norms type collagen (100μg/ml, 24 h, 4° C.), and 20 μl of 1 M Ca2+ was added to initiateclotting. Samples (10 μl) were added to the wells of a microtiter platecontaining 90 μl of 3.8% sodium citrate at 2.5-10 min intervals for 60min. Color was developed for 2 min by the addition of 50 l of 2mmol/liter S-2238 (H-D-Phe-Arg-NH-NO2-HCl, a thrombin-specificsubstrate; Chromogenix, Mölndal, Sweden) in 1 mol/liter Tris (pH 8.1).The absorbance of the released color product was measuredspectrophotometrically at a wavelength of 405 nm using a Vmax microtiterplate reader (Easy Reader, EAR 340AT, SLT Lab Instruments GmbH, Vienna,Austria). Measurements were obtained in triplicate at each time point.

Statistical Evaluation

Statistical analysis was performed using the unpaired Student's t test.

Although only preferred embodiments are specifically illustrated anddescribed herein, it will be appreciated that many modifications andvariations of the present invention are possible in light of the aboveteachings and within the purview of the appended claims withoutdeparting from the spirit and intended scope of the invention.

1.-16. (canceled)
 17. A method of treating a patient at risk for theformation and/or the stabilization of thrombi comprising administeringto said patient at least one inhibitor of Factor XII or activated FactorXII (collectively FXII), wherein the patient has, has had, or is at riskfor: venous thrombosis, arterial thrombosis, stroke, myocardialinfarction, or use of a prosthetic device, and wherein the at least oneinhibitor of FXII is not ATIII inhibitor, angiotensin converting enzymeinhibitor, C1 inhibitor, aprotinin, alpha-1 protease inhibitor,leupeptin, ecotin, bovine pancreatic trypsin inhibitor mutants,antipain, or DX88.
 18. The method of claim 17, wherein the at least oneinhibitor is at least one protease inhibitor.
 19. The method of claim18, wherein the at least one protease inhibitor is a serine proteaseinhibitor.
 20. The method of claim 18, wherein the at least one proteaseinhibitor is Z-Pro-Pro-aldehyde-dimethyl acetate.
 21. The method ofclaim 18, wherein the at least one protease inhibitor is chosen from oneor more of: Fmoc-Ala-Pyr-CN, corn-trypsin inhibitor, yellowfin soleanticoagulant protein, Curcurbita maxima trypsin inhibitor-V, andCurcurbita maxima isoinhibitor.
 22. The method of claim 17, wherein theprosthetic device is a haemodialyser, cardiopulmonary by-pass circuit,vascular stent, or in-dwelling catheter.
 23. The method of claim 17,wherein the patient has, has had, or is at risk for venous thrombosis.24. The method of claim 17, wherein the patient has, has had, or is atrisk for arterial thrombosis.
 25. The method of claim 17, wherein thepatient has, has had, or is at risk for stroke.
 26. The method of claim17, wherein the patient has, has had, or is at risk for myocardialinfarction.